1. vnd/NK-2 Homeobox Gene Promoter Regions. [unreadable] [unreadable] Previous studies from this laboratory showed that the vnd/NK-2 gene is expressed in 11 neuroblasts per hemisegment in Drosophila embryos. A -5.3 to -2.8 kb DNA fragment upstream of the vnd/NK-2 transcription start site is required for the expression of the vnd/NK-2 gene in ten of the eleven neuroblasts. To identify nucleotide sequences that regulate vnd/NK-2 expression in more detail the nucleotide sequences of the 5-prime-upstream regions of the vnd/NK-2 genes from seven species of Drosophila that originated between three and forty million years ago were compared. Small conserved nucleotide sequences were found that are candidates for nucleotide sequences that regulate the expression of the vnd/NK-2 gene. DNA constructs of the vnd/NK-2 5-prime-upstream region of the vnd/NK-2 gene were prepared, each with a small deletion of a highly conserved nucleotide sequence. Five or more transgenic fly lines for each deleted motif were generated and the expression pattern of the vnd/NK-2 gene was analyzed by immunohistochemistry. Nucleotide sequences were identified that regulate the expression of the vnd/NK-2 gene in neuroblasts.[unreadable] [unreadable] 2. Synaptogenesis in Development and Disease: Analysis of Drosophila Neuroligin Genes.[unreadable] [unreadable] In vertebrates neuroligin proteins are post-synaptic cell-adhesion molecules that bind to a pre-synaptic protein, beta-neurexin, thereby inducing the formation of a class of glutaminergic synapses. Thus neuroligin/neurexin interactions play an important role in synapse formation and the specification of neural circuits in the brain. Knock-out mutations of mouse neuroligin genes have not been described; however, mutations in human neuroligins have been found that are associated with autism and mental retardation. Thus, the study of Drosophila neuroligins affords opportunities to apply genetic and behavioral tools to study the formation of synapses, neural circuits, and behavior. [unreadable] [unreadable] The developmental profiles of expression of the four Drosophila neuroligin genes were determined by RT-PCR using RNA samples from adult head, larval brain, and 0-16 hour embryos. All four genes were detected in adult head as well as in third instar larval brain/CNS. Only two neuroligins were detected in embryos. The CG31146 gene was shown to be expressed in the CNS of Drosophila larvae. Expression of this gene also was detected in subsets of neurons in the adult brain.[unreadable] [unreadable] To determine the functions of neuroligin genes Nrlg1 and CG31146, loss-of-function mutations were generated. Nrlg1-deltaxb13 is a 33 kb deletion that removes the entire coding region and 21 kb of DNA 5-prime from the transcription start site. Flies homozygous for this null allele are viable and fertile and do not display any obvious behavioral phenotypes.[unreadable] [unreadable] CG31146-delta46-24 is a deletion of 30 kb that removes 10 kb of DNA 5-prime from the transcription start site and part of the coding sequence, including the first ten exons. Flies homozygous for this allele are viable and fertile, but appear sluggish and less active than wild-type flies. Although both homozygous mutants are viable, the double homozygous mutant combination resulted in lethality, which suggests an overlap of essential functions for the two genes.[unreadable] [unreadable] [unreadable] 3. Identification of Molecules that Enhance Memory.[unreadable] [unreadable] CREB is a transcription factor whose activation enhances some types of long-term memory. The goal of this project is to screen a large library of compounds in a high throughput format to find compounds that enhance CREB activation mediated by partial activation of adenylyl cyclase. Compounds found are candidate compounds that may stimulate the establishment of certain types of long-term memory. A cell line stably transfected with a vector that contains CRE sites linked to a reporter gene was used. Thus far, more than seventy thousand compounds were tested using many concentrations of each compound and hundreds of compounds active at low concentrations, were found. Candidate compounds will be subjected to further tests to reduce the number of compounds that will be tested for their effects on long-term memory.[unreadable] [unreadable] 4. Longitudinals Lacking (lola) Gene.[unreadable] [unreadable] The lola gene encodes a family of C2H2 type zinc finger protein transcription factors that are involved in axon guidance and development of the nervous system. Many different alternatively-spliced isoforms of lola mRNA are expressed. Seventeen species of lola mRNA encode lola proteins that have identical N-terminal regions, but have different pairs of zinc fingers in the C-terminal regions. The hypothesis that different isoforms of lola protein that contain different pairs of zinc fingers regulate different sets of genes was tested. Double-stranded RNAs encoding the variable C-terminal regions of the seventeen isoforms of lola mRNA were synthesized and each RNA preparation was injected into early Drosophila embryos to decrease the level of the corresponding species of lola mRNA by RNA interference. Embryos were incubated to allow development to proceed. Then RNA from each set of injected embryos was isolated, labeled and hybridized to Drosophila nucleic acid microarray chips capable of revealing mRNA levels of almost all of the 14,300 genes in the Drosophila genome. The results show that some of the isoforms of lola mRNA encode proteins that regulate different sets of genes. During the past year, much work has focused on analysis of the data, which involves hundreds of genes, and preparing a manuscript.[unreadable] [unreadable] 5. Genes Expressed by Undifferentiated Versus Differentiated Drosophila Neural Cells.[unreadable] [unreadable] Conditions were found to shift cells from a Drosophila neural cell line from a relatively undifferentiated state to a differentiated state. RNA was prepared from cells at different times during the differentiation process. Drosophila S2 cells (a non-neural cell line) were treated in exactly the same manner and RNA also was prepared at different times during the treatment process. About 250 genes were identified whose expression changed during differentiation only in the neural cells. Another set of genes was identified whose expression changed both in neural cells and non-neural S2 cells, and a third set of genes was identified whose expression changed only in the non-neural S2 cells. A manuscript currently is being prepared.[unreadable] [unreadable] 6. Genes That Affect the Assembly of the Nervous System in Drosophila Embryos.[unreadable] [unreadable] In previous studies from this laboratory, RNA interference was used to screen approximately 5,400 Drosophila double-stranded RNAs and 43 genes were found that affect the structure of the embryonic nervous system when the levels of the corresponding species of mRNA were decreased by RNAi. During the past year approximately 1,000 additional double-stranded RNAs were screened, which, together with the previous double-stranded RNAs tested, correspond to approximately 45% of the Drosophila genome. Twelve additional genes were found whose mRNAs are required for the formation of the nervous system in embryos.[unreadable] [unreadable] Note: Three manuscripts are in preparation for projects 4, 5, and 6.